Solving the enigma: Mass spectrometry and small molecule probes to study sphingolipid function

Sphingolipids are highly bioactive lipids. Sphingolipid metabolism produces key membrane components (e.g. sphingomyelin) and a variety of signaling lipids with different biological functions (e.g. ceramide, sphingosine-1-phosphate). The coordinated activity of tens of different enzymes maintains proper levels and localization of these lipids with key roles in cellular processes. In this review, we highlight the signaling roles of sphingolipids in cell death and survival. We discuss recent findings on the role of specific sphingolipids during these processes, enabled by the use of lipidomics to study compositional and spatial regulation of these lipids and synthetic sphingolipid probes to study subcellular localization and interaction partners of sphingolipids to understand the function of these lipids.     

Loss of alkaline ceramidase inhibits autophagy in Arabidopsis and plays an important role during environmental stress response

Sphingolipids, a class of bioactive lipids found in cell membranes, can modulate the biophysical properties of the membranes and play a critical role in signal transduction. Sphingolipids are involved in autophagy in humans and yeast, but their role in autophagy in plants is not well understood. In this study, we reported that the AtACER, an alkaline ceramidase that hydrolyses ceramide to long‐chain base (LCB), functions in autophagy process in Arabidopsis. Our empirical data showed that the loss of AtACER inhibited autophagy, and its overexpression promoted autophagy under nutrient, salinity, and oxidative stresses. Interestingly, nitrogen deprivation significantly affected the sphingolipid's profile in Arabidopsis thaliana, especially the LCBs. Furthermore, the exogenous application of LCBs also induced autophagy. Our findings revealed a novel function of AtACER, where it was found to involve in the autophagy process, thus, playing a crucial role in the maintenance of a dynamic loop between sphingolipids and autophagy for cellular homeostasis under various environmental stresses.     

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to “switch” autophagy toward cell survival (e.g., sphingosine-1-phosphate) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the “switch” between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.     

Sphingolipids in mitochondria

Sphingolipids are bioactive lipids found in cell membranes that exert a critical role in signal transduction. In recent years, it has become apparent that sphingolipids participate in growth, senescence, differentiation and apoptosis. The anabolism and catabolism of sphingolipids occur in discrete subcellular locations and consist of a strictly regulated and interconnected network, with ceramide as the central hub. Altered sphingolipid metabolism is linked to several human diseases. Hence, an advanced knowledge of how and where sphingolipids are metabolized is of paramount importance in order to understand the role of sphingolipids in cellular functions. In this review, we provide an overview of sphingolipid metabolism. We focus on the distinct pathways of ceramide synthesis, highlighting the mitochondrial ceramide generation, transport of ceramide to mitochondria and its role in the regulation of mitochondrial-mediated apoptosis, mitophagy and implications to disease. We will discuss unanswered questions and exciting future directions. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases.     

Ceramide and mitochondria in ischemic brain injury

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation and apoptosis. Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo at the cytosolic side of the endoplasmic reticulum serving as precursors for the biosynthesis of sphingolipids in the Golgi. Recently, mitochondria emerged as an important intracellular compartment of sphingolipid metabolism. Thus, several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase, novel neutral sphingomyelinase, and (dihydro) ceramide synthase, an important ceramide-generating enzyme in de novo ceramide synthesis and recycling pathway. Mitochondrial dysfunction appears to be essential in tissue damage after brain ischemia/reperfusion (IR). Mitochondria are known to be involved in both the necrosis and apoptosis detected in animal models of ischemic stroke, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function. Although mitochondrial injury in stroke has been extensively studied and key mitochondrial functions affected by IR are mainly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. Emerging data indicate a deregulation of ceramide metabolism in mitochondria damaged by IR suggesting that ceramides could play critical roles in cerebral IR-induced mitochondrial damage. This review will examine the experimental evidence supporting the key role of ceramides in mitochondrial dysfunction in cerebral IR and highlight potential targets for development of novel therapeutic approaches for stroke treatment.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp^−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.     

Sphingolipid depletion impairs endocytic traffic and inhibits Wingless signaling

Sphingolipids are an important part of the plasma membrane and implicated in a multitude of cellular processes. However, little is known about the role of sphingolipids in an epithelial context and their potential influence on the activity of signaling pathways. To shed light on these aspects we analyzed the consequences of changing ceramide levels in vivo in the Drosophila wing disc: an epithelial tissue in which the most fundamental signaling pathways, including the Wnt/Wg signaling pathway, are well characterized.We found that downregulation of Drosophila’s only ceramide synthase gene schlank led to defects in the endosomal trafficking of proteins. One of the affected proteins is the Wnt ligand Wingless (Wg) that accumulated. Unexpectedly, although Wg protein levels were raised, signaling activity of the Wg pathway was impaired. Recent work has spotlighted the central role of the endocytic trafficking in the transduction of the Wnt signal. Our results underscore this and support the view that sphingolipid levels are crucial in orchestrating epithelial endocytic trafficking in vivo. They further demonstrate that ceramide/sphingolipid levels can affect Wnt signaling.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

LRRK2 Transport Is Regulated by Its Novel Interacting Partner Rab32

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson''s disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.     

Roles and regulation of secretory and lysosomal acid sphingomyelinase

Acid sphingomyelinase occupies a prominent position in sphingolipid catabolism, catalyzing the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Enzymatic dysfunction of acid sphingomyelinase results in Niemann–Pick disease, a lysosomal storage disorder characterized at the cellular level by accumulation of sphingomyelin within the endo-lysosomal compartment. Over the past decade interest in the role of acid sphingomyelinase has moved beyond its ‘housekeeping’ function in constitutive turnover of sphingomyelin in the lysosome to include study of regulated ceramide generation. Ceramide functions as a bioactive sphingolipid with pleiotropic signaling properties, and has been implicated in diverse cellular processes of physiologic and pathophysiologic importance. Though many cellular enzymes have the capacity to generate ceramide, there is growing appreciation that ‘all ceramides are not created equal.’ Ceramides likely exert distinct effects in different cellular/subcellular compartments by virtue of access to other sphingolipid enzymes (e.g. ceramidases), effector molecules (e.g. ceramide-activated protein phosphatases), and neighboring lipids and proteins (e.g. cholesterol, ion channels). One of the unique features of acid sphingomyelinase is that it has been implicated in the hydrolysis of sphingomyelin in three different settings — the endo-lysosomal compartment, the outer leaflet of the plasma membrane, and lipoproteins. How a single gene product has the capacity to function in these diverse settings, and the subsequent impact on downstream ceramide-mediated biology is the subject of this review.     

The Endo-lysosomal System in Parkinson’s Disease: Expanding the Horizon

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease, and its prevalence is increasing with age. A wealth of genetic evidence indicates that the endo-lysosomal system is a major pathway driving PD pathogenesis with a growing number of genes encoding endo-lysosomal proteins identified as risk factors for PD, making it a promising target for therapeutic intervention. However, detailed knowledge and understanding of the molecular mechanisms linking these genes to the disease are available for only a handful of them (e.g. LRRK2, GBA1, VPS35). Taking on the challenge of studying poorly characterized genes and proteins can be daunting, due to the limited availability of tools and knowledge from previous literature. This review aims at providing a valuable source of molecular and cellular insights into the biology of lesser-studied PD-linked endo-lysosomal genes, to help and encourage researchers in filling the knowledge gap around these less popular genetic players. Specific endo-lysosomal pathways discussed range from endocytosis, sorting, and vesicular trafficking to the regulation of membrane lipids of these membrane-bound organelles and the specific enzymatic activities they contain. We also provide perspectives on future challenges that the community needs to tackle and propose approaches to move forward in our understanding of these poorly studied endo-lysosomal genes. This will help harness their potential in designing innovative and efficient treatments to ultimately re-establish neuronal homeostasis in PD but also other diseases involving endo-lysosomal dysfunction.     

Widespread tissue distribution and synthetic pathway of polyunsaturated C24:2 sphingolipids in mammals

Sphingolipids are multifunctional lipids and a major constituent of the cell membranes of eukaryotes. Although the fatty acid (FA) moiety of sphingolipids is usually a saturated or monounsaturated FA, polyunsaturated FA (PUFA)-containing species also exist in mammalian tissues. In the present study, we showed that C24:2 PUFA-containing ceramide is one of the seven major ceramide species in a wide range of tissues. C24:2 ceramide levels were especially high in spleen and small intestine; in the former, it was the fourth most abundant ceramide species. However, both the synthetic pathway and the physiological function of C24:2 ceramide had yet to be identified. Tracer analysis using deuterium-labeled linoleic acid (C18:2) revealed that C24:2 ceramide is produced via elongation of linoleic acid. We also found that the FA elongase ELOVL1 and the ceramide synthase CERS2 were involved in C24:2 ceramide production. Sphingolipids are known to form lipid microdomains in membranes; however, in a detergent-resistant membrane (DRM) assay, we observed a lower proportion of C24:2 sphingomyelin in the DRM fraction than of saturated sphingomyelins, suggesting that C24:2 sphingolipids may act to negatively regulate lipid microdomain formation. Our findings expand our knowledge of sphingolipid diversity, and provide insight into how different sphingolipid molecular species play different functions in biological membranes.     

Autophagy regulates sphingolipid levels in the liver

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.     

A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

On the nature of ceramide-mitochondria interactions – Dissection using comprehensive mitochondrial phenotyping

Sphingolipids are a unique class of lipids owing to their non-glycerol-containing backbone, ceramide, that is constructed from a long-chain aliphatic amino alcohol, sphinganine, to which a fatty acid is attached via an amide bond. Ceramide plays a star role in the initiation of apoptosis by virtue of its interactions with mitochondria, a control point for a downstream array of signaling cascades culminating in apoptosis. Many pathways converge on mitochondria to elicit mitochondrial outer membrane permeabilization (MOMP), a step that corrupts bioenergetic service. Although much is known regarding ceramides interaction with mitochondria and the ensuing cell signal transduction cascades, how ceramide impacts the elements of mitochondrial bioenergetic function is poorly understood. The objective of this review is to introduce the reader to sphingolipid metabolism, present a snapshot of mitochondrial respiration, elaborate on ceramides convergence on mitochondria and the upstream players that collaborate to elicit MOMP, and introduce a mitochondrial phenotyping platform that can be of utility in dissecting the fine-points of ceramide impact on cellular bioenergetics.     

Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and sphingosine 1-phosphate (S1P) constitute a rheostat system in which ceramide promotes cell death and S1P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S1P, respectively.     

Anti-apoptotic and anti-oxidative mechanisms of minocycline against sphingomyelinase/ceramide neurotoxicity: implication in Alzheimer's disease and cerebral ischemia

Sphingolipids represent a major class of lipids in which selected family members act as bioactive molecules that control diverse cellular processes, such as proliferation, differentiation, growth, senescence, migration and apoptosis. Emerging evidence reveals that sphingomyelinase/ceramide pathway plays a pivotal role in neurodegenerative diseases that involve mitochondrial dysfunction, oxidative stress and apoptosis. Minocycline, a semi-synthetic second-generation tetracycline derivative in clinical use for infection control, is also considered an effective protective agent in various neurodegenerative diseases in pre-clinical studies. Acting via multiple mechanisms, including anti-inflammatory, anti-oxidative and anti-apoptotic effects, minocycline is a desirable candidate for clinical trials in both acute brain injury as well as chronic neurodegenerative disorders. This review is focused on the anti-apoptotic and anti-oxidative mechanisms of minocycline against neurotoxicity induced by sphingomyelinase/ceramide in relation to neurodegeneration, particularly Alzheimer's disease and cerebral ischemia.